水华蓝藻产毒特性的PCR检测法

特异引物对（ＴＯＸ　１Ｐ／１Ｆ；ＴＯＸ　２Ｐ／２Ｆ）用于检测微囊藻毒素合成酶基因ｍｃｙＢ片段在３８种水华蓝藻中的分布情况。结果显示，所有能产生微囊藻毒素的微囊藻都有特异扩增条带，非产毒株则没有。几种常规的毒性检测方法验证了ＰＣＲ方法所获结果的准确性。本研究发展了以全细胞ＰＣＲ法检测ｍｃｙＢ片断，说明全细胞ＰＣＲ检测法适用于不同来源的蓝藻材料。结果证明以ＤＮＡ为基础鉴别产毒和非产毒微囊藻及其他水华蓝藻的方法是可行和实用的。

Allelopathic mechanism of pyrogallol to Microcystis aeruginosa PCC7806 (Cyanobacteria): From views of gene expression and antioxidant system

Pyrogallol is a potent allelochemical on Microcystis aeruginosa, but its allelopathic mechanism is not fully known. In order to explore this mechanism, gene expressions for prx, mcyB, psbA, recA, grpE, fabZ under pyrogallol stress were studied, and activities of the main antioxidant enzymes were also measured. The results showed that expression of grpE and recA showed no significant change under pyrogallol stress, while psbA and mcyB were up-regulated at 4 mg L-1. Both prx and fabZ were up-regulated even under exposure to 1 mg L-1 pyrogallol concentration. The activities of superoxide dismutase (SOD) and catalase (CAT) were enhanced under pyrogallol stress. Levels of malodialdehyde (MDA) at 2 and 4 mg L-1 pyrogallol were significantly higher than those of the controls. It was concluded that oxidant damage is an important mechanism for the allelopathic effect of pyrogallol on M. aeruginosa. (c) 2009 Elsevier Ltd. All rights reserved.

Detection of hepatotoxic Microcystis strains by PCR with intact cells from both culture and environmental samples

Microcystins are small hepatotoxic peptides produced by a number of cyanobacteria. They are synthesized non-ribosomally by multifunctional enzyme complex synthetases encoded by the mcy genes. Primers deduced from mcy genes were designed to discriminate between toxic microcystin-producing strains and non-toxic strains. Thus, PCR-mediated detection of mcy genes could be a simple and efficient means to identify potentially harmful genotypes among cyanobacterial populations in bodies of water. We surveyed the distribution of the mcyB gene in different Microcystis strains isolated from Chinese bodies of water and confirmed that PCR can be reliably used to identify toxic strains. By omitting any DNA purification steps, the modified PCR protocol can greatly simplify the process. Cyanobacterial cells enriched from cultures, field samples, or even sediment samples could be used in the PCR assay. This method proved sensitive enough to detect mcyB genes in samples with less than 2,000 Microcystis cells per ml. Its accuracy, specificity and applicability were confirmed by sequencing selected DNA amplicons, as well as by HPLC, ELISA and mouse bioassay as controls for toxin production of every strain used.

Microcystin-producing genotypes from cyanobacteria in Brazilian reservoirs

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Detecção molecular quantitativa de cianobactérias hepatotóxicas através de PCR competitiva

Pós-graduação em Ciências Biológicas (Biologia Vegetal) - IBRC

Microcystin-producing genotypes from cyanobacteria in Brazilian reservoirs

The aim of this study was to evaluate the use of new oligonucleotide primers (mcyB-F/R, mcyB-F/R-A, and mcyB-F/R-B) designed from Brazilian cyanobacteria for the detection of microcystin-producing genotypes in 27 environmental samples from water reservoirs and 11 strains of Microcystis. Microcystins were found using HPLC in all 11 strains and 19 of the environmental samples. The new oligonucleotide primers amplified fragments of microcystin-producing genes, including the eight environmental samples in which no microcystins were detected by HPLC, but which presented amplified fragments, thereby demonstrating the existence of microcystin-producing genes. The new oligonucleotide primers exhibited better specificity when used with environmental samples and were more reliable in comparison with those described in the literature (mcyB-FAA/RAA and mcyA-Cd/FR), which generate false-negative results. The better performance of these new oligonucleotide primers underline the need for designing molecular markers that are well fitted to the regional biological diversity. As this is a fast predictive technique for determining the presence or absence of microcystins, it could be used either alone or in conjunction with other techniques, such as the screening of samples to be sent for quantitative toxicological analysis using HPLC, thereby reducing monitoring cost and time. (c) 2010 Wiley Periodicals, Inc. Environ Toxicol, 2012.